JOURNAL OF VmoLOwy, Apr. 1976, P. 156-166

Vol. 18, No. 1 Printed in U.SA.

Copyright © 1975 American Society for Microbiology

Viral Transcription in KB Cells Infected by TemperatureSensitive "Early" Mutants of Adenovirus Type 5 TIMOTHY H. CARTER* AND HAROLD S. GINSBERG Department of Microbiology, College of Physicians and Surgeons, Columbia University, New York, New York 10032 Received for publication 21 October 1975

RNA:DNA hybridization was used to study the synthesis of viral RNA in two DNA-minus, temperature-sensitive mutants of type 5 adenovirus (H5ts125 and H5ts149) belonging to two different, non-overlapping complementation groups. Hybridization competition analysis showed that both mutants transcribed all early gene sequences at the restrictive temperature (41 C). In mutant-infected cells at 41 C, the rate of viral transcription was similar to the rate of early RNA synthesis in wild-type virus infection and was dependent on the multiplicity of infection; little or no late transcription was detected. The shutoff of class I early RNA transcription was shown to be a late function during wild-type virus infection and did not occur at 41 C in mutant-infected cells. When mutant-infected cells were incubated at the permissive temperature (32 C) for 25 h and then shifted to 41 C, the rate of viral DNA synthesis decreased rapidly for H5ts125 and slowly for H5ts149. However, the rate of viral transcription remained unchanged in H5ts125-infected cells for at least 3 h after the temperature shift; although the synthesis of viral DNA had stopped by this time, the synthesis of late viral RNA sequences continued. After type 2 or 5 adenovirus infection of KB cells, early viral transcription can be detected 2 h later and represents only a small fraction of the total RNA synthesized in infected cells (9, 12). The late phase of viral transcription begins with a dramatic increase in the rate of synthesis of virus-specific RNA coincident with the replication of viral DNA (1, 12). The predominant genetic sequences transcribed at this time consist of a subset of the early viral genes (class II; reference 12) and a new group of genes that occupies a major fraction of the viral genome (6, 9, 12). The remaining early genes (class I; reference 12) are transcribed much less frequently, if at all, after DNA synthesis has begun (6, 12). The central role played by viral DNA replication in the regulation of the transcriptional program of adenovirus has been inferred from experiments in which DNA replication was prevented by inhibitors such as arabinosyl cytosine (araC) (12), 5-fluorodeoxyuridine (1), and cycloheximide (5, 14). In each case, early transcription occurred normally, and inhibitors prevented the shift to late gene expression. The availability of temperature-sensitive mutants of type 5 adenovirus (AdS) now enables the study of genetic regulation without resort to inhibitors. Two unique temperaturesensitive mutants of Ad5, H5ts125 (ts125) and

H5ts149 (ts149), which are unable to replicate their DNA at the restrictive temperature, have previously been described (7, 10). The present communication summarizes studies using mutant-infected cells to characterize viral transcription in cells and describes the use of these mutants to study the relationship between the replication of viral DNA and the regulation of early and late viral RNA synthesis.

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MATERIALS AND METHODS Cell and virus growth. Suspension cultures of KB cells were grown, and viral stocks were prepared and titrated by immunofluorescence, as described in previous communications (8, 12). Infection of cells for early and late viral RNA. Spinner cultures of KB cells at a concentration of 1.5 x 105/ml were infected with 100 to 200 PFU of wild-type (wt) adenovirus per cell at 36 C (12). For the isolation of early RNA, 50 AiM araC was added 1 h after infection, and the cells were harvested at 7 h. Labeled early RNA was obtained by addition of [3H]uridine 2 h after infection. Late viral RNA was isolated from cells infected for 19 h in the absence of araC; to label late RNA, [3H]uridine was added for the last hour of infection. Infected cells were harvested by centrifugation at 4 C, washed once with ice-cold phosphate-buffered saline, and stored at -20 C until used. Radioactive labeling of viral RNA and DNA. Infected-cell DNA was pulse labeled from either 20 min or 1 h with [ methyl-3H]thymidine (specific

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TRANSCRIPTION BY Ad5 ts EARLY MUTANTS

activity, 40 to 60 Ci/mmol) at 1.25 ACi/ml. To label virion DNA, [3H]thymidine or [methyl- 4C]thymidine (specific activity, 40 to 60 mCi/mmol), 25 or 0.1 ;Ci/ml, respectively, was added to cultures 9 to 40 h after infection. Viral RNA was labeled in spinner cultures with [3,5-3H]uridine (specific activity, 40 to 60 Ci/mmol) at 2 to 5 MCi/ml. In experiments at 41 C, labeling was terminated by addition of NaN3 to a final concentration of 20 mM and centrifugation at 4 C. Extraction of DNA. DNA was extracted from purified virus as previously described (1). DNA from infected cells was extracted as follows: 1.5 x 107 to 3.0 x 107 cells were suspended in 2 ml of buffer containing 10 mM sodium acetate, pH 5.2, and 0.14 M NaCl. To lyse cells, sodium dodecyl sulfate (SDS) was added to a final concentration of 1%. The suspension was briefly sonicated to shear the DNA and was then extracted twice with an equal volume of distilled phenol, which had been saturated with lysis buffer. The extracted nucleic acids were precipitated at -20 C by addition of 3 to 5 volumes of ethanol. The precipitate was collected by centrifugation, washed once with cold 70% ethanol, dissolved in 1 ml of buffer containing 10 mM Trishydrochloride, pH 7.4, 0.1 M NaCl, and 10 mM MgCl2, and incubated for 30 min at 37 C with 20 tg of pancreatic RNase per ml that had been heated to 80 C for 10 min to inactivate contaminating DNase. SDS was then added to a concentration of 0.5%, followed by incubation with 250 Ag of predigested Pronase (19) per ml for 30 min at 37 C. The digest was then extracted twice with water-saturated phenol, and the DNA was precipitated with ethanol as before. Between 100 and 250 ,ug of DNA was usually recovered from 107 cells. The concentration of DNA was measured spectrophotometrically at 260 nm, using an extinction coefficient of 0.02 ug-1 ml-'. Extraction of RNA. RNA was extracted from whole infected cells by a modification of the hot phenol-SDS procedure used by Lucas and Ginsberg (12). The protocol was identical to the protocol for DNA extraction with the following exceptions: sonication of the lysate was omitted; the first phenol extraction was done at 65 C; and nuclease digestion used 10 ;g of electrophoretically purified DNase per ml. From 3 to 7 mg of RNA was recovered from 1.5 x 108 cells. Preparation of DNA membrane filters. DNA was denatured in 0.1 M NaOH (2) and immobilized on cellulose nitrate membrane filters (B6, 25-mm diameter; Schleicher and Schuell, Keene, N. H.) as described previously (12). After binding DNA to 25-mm filters, 6.5-mm-diameter filters were cut with a paper punch. Hybridization of RNA to filter-bound DNA. Filters (6.5 mm) with Ad5 DNA were incubated with labeled RNA in 0.1 to 0.2 ml of buffer containing 30% formamide (purified according to Tibbitts et al. [18]), 10 mM Tris-hydrochloride, pH 7.4, 0.45 M NaCl, 10 mM EDTA, and 0.1% SDS at 45 C (5) for 48 h. Filters were processed by washing three times with 2x SSC (SSC is 0.15 M NaCl and 0.015 M sodium citrate), treating for 1 h at room temperature

157

with 20 ug of pancreatic RNase per ml (heated as above) in 2x SSC, and washing three more times with 2x SSC. Radioactivity bound to the dried filters was measured in a liquid scintillation counter in a toluene-based scintillator. Total labeled viral RNA was measured by hybridization to a series of filters containing 0.5, 1.0, 2.0, and 4.0 ,g of Ad5 DNA, and maximum hybridization (at infinite DNA concentration) was calculated according to Lucas and Ginsberg (12). Two-stage hybridization competition analysis (12) was carried out as follows. In the first stage, increasing amounts of unlabeled competitor RNA, which had been partially hydrolyzed in 1 M NaOH at room temperature for 90 s, were incubated for 4 days with filters containing 0.05 or 0.5 ,ug of denatured Ad5 DNA (2, 12). In the second stage the filters were washed three times with 2x SSC and incubated for 48 h with 3H-labeled RNA. After the second incubation, the filters were washed and treated with RNase and the radioactivity was determined as described above. Hybridization of DNA to filter-bound viral DNA. Approximately 100 ,ug of 3H-labeled DNA extracted from infected cells was mixed with a constant amount of purified 14C-labeled virion DNA (0.1 to 1.0 Mg) in 0.lx SSC and boiled for 30 min. The denatured DNA solution was cooled rapidly in an ice bath and mixed with an equal volume of buffer containing 0.6 M NaCl, 20 mM HEPES, pH 7.0 (HEPES is N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid), 20 mM EDTA, and 0.4% SDS. A sample was counted to determine the total radioactivity, and the remainder was added to a scintillation vial containing two 25-mm filters, one loaded with 5 or 10 ug of Ad5 DNA and the other loaded with the same amount of Xdlac DNA. The reactions were overlayered with mineral oil and incubated at 65 C for 20 h. The filters were then washed three times on each side with 10-ml volumes of 2 x SSC and treated with S1 endonuclease (16) at 55 C for 2 h in 2 ml of 0.3 M NaCl, 30 mM sodium acetate, pH 4.5, 4 mM ZnSO4, and 25 Aig of heat-denatured salmon sperm DNA per ml. The filters were then washed as before, dried, and counted in a toluenebased scintillator. The counts hybridized to Xdlac DNA were subtracted from the counts hybridized to Ad5 DNA, and the efficiency of hybridization was determined from the fraction of "4C-labeled virion DNA bound to the Ad5 DNA filter. This efficiency, usually 15 to 60% in the range of DNA concentrations used, was used to correct the 3H hybridization. Reconstruction experiments in which increasing amounts of 3H-labeled DNA from purified AdS were hybridized in the presence of a constant amount of "4C-labeled adenovirus DNA gave a linear relationship between the corrected hybridization of purified 3H-labeled viral DNA and the 3H-labeled DNA input (see Appendix). Radioisotopes. [3,5-3H]uridine and [methyl-3H]and [methyl-'4C]thymidine were purchased at highest available specific activities from Schwarz-Mann (Orangeburg, N.Y.). Enzymes and reagents. Pancreatic RNase (grade R), electrophoretically purified DNase I (grade

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DPFF), and T1 RNase were obtained from Worthington Biochemicals Corp. (Freehold, N.J.). Pronase (grade B) was purchased from Calbiochem (La Jolla, Calif.). Endonuclease S1 was purified from Taka Diastase by the method of Sutton (16). araC was purchased from Calbiochem, and HEPES buffer was purchased from Sigma Chemical Co. (St. Louis, Mo.).

RESULTS Rate of viral RNA synthesis at the restrictive temperature. The rate of viral RNA synthesis in cells infected at a restrictive temperature (39.5 C) with ts125, ts149, or wt virus was measured by hybridization of pulse-labeled RNA to increasing amounts of viral DNA as described in Materials and Methods (Fig. 1). When wt virus-infected cells were labeled for 20-min periods, virus-specific RNA synthesis was detected at 2 h after infection. The rate of viral RNA synthesis increased after 6 h and by Inn1). 0

z

0

0

10ot

10

0 z

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1.0o

cr I

CD

0.1i

-

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C-)

0 H-

0.01I 0

5

10

15

20

25

HOURS p.i. FIG. 1. Rate of viral RNA synthesis at 39.5 C. KB cells in spinner culture were grown at 36 C and equilibrated at 39.5 C for 1 h before infection with 50 PFU of Ad5 wt (0), H5ts125 (0), or H5ts149 (A) per cell. Samples (100 ml) were pulse labeled with 0.5 mCi of [3H]uridine for 30 min at the indicated times. RNA was extracted from each sample and hybridized to a set of Ad5 DNA filters to determine the total amount of radioactive viral RNA, as described in the text. The results of two separate experiments are shown: Ad5 wt and ts125 were analyzed in the same experiment, and ts149 was analyzed in a second experiment.

15 h was nearly 300 times that observed at 3 h. In contrast, when either mutant was used to infect cells, no large change in rate of viral RNA synthesis was detected. However, a small, continuous increase in viral transcription was consistently observed in ts125-infected cells starting about 10 h after infection. To ascertain whether the small increase in viral RNA synthesis in ts125-infected cells at 39.5 C was the result of leakiness of the DNAminus mutant, the effects of temperature and multiplicity of infection on the rates of viral transcription and DNA synthesis were tested. The restrictive temperature was increased to 41 C on the assumption that a more stringent restrictive condition might abolish or reduce any residual mutant function expressed at 39.5 C. In addition, duplicate cultures were infected at low and high multiplicities (5 and 50). If a threshold concentration of mutant protein is necessary to produce the effect seen with ts125, the effect should be more pronounced at higher multiplicities of infecting virus. The results shown in Table 1 indicate that with either multiplicity, and even at the more restrictive temperature, the rate of viral transcription in ts125-infected cells was greater at 15 than at 5 h after infection. Since the rate of viral DNA synthesis at 13.5 h was less than 0.5% of wt, this mutant was not leaky for DNA synthesis at 41 C. A small amount of incorporation of label into viral DNA was reproducibly detected in ts149-infected cells. Although this incorporation amounted to only about 20% more than the background level in mock-infected cells (Table 1), ts149 may be slightly leaky for DNA synthesis even at 41 C. Alternatively, this incorporation of thymidine could have been the result of slightly increased repair activity in this mutant. Synthesis of early viral RNA. To characterize the viral RNA sequences present in cells infected by the mutants at the restrictive temperature, whole-cell RNA was extracted 15 h after infection and used to compete with the hybridization of labeled wt early RNA (Fig. 2). The complete inhibition of wt early RNA hybridization by RNA from mutant-infected cells indicated that all early sequences were synthesized by both mutants at 41 C and were still present in the cells 15 h after infection. Shutoff of class I early RNA synthesis. Although in wt infection the synthesis of class I early viral RNA stops during the early phase of viral DNA synthesis (12), it has not been demonstrated that the shutoff is a late gene function. To test for the shutoff of class I mRNA synthesis in the absence of viral DNA replication, and hence in the absence of late

TRANSCRIPTION BY Ad5 ts EARLY MUTANTS

VOL. 18, 1976

TABLE 1. Viral DNA and RNA synthesis at 41 Ca Virus strain PFU/cell

Early RNAb

Late

R a RNA

DN DNA

9.1 37.2

21.2 50.1

wt

5 50

0.09 0.63

ts125

5 50

0.05 0.28

0.12 0.50

0.14 0.25

ts149

5 50

0.03 0.53

0.10 NDc

0.51 ND

0

0.004

Nd

Mock

0.41

Spinner cultures were infected at 41 C with 5 or 50 PFU of Ad5 wt, ts125, or ts149 per cell. One culture was not infected (mock). Samples (50 ml) were labeled for 1 h with 750 pCi of [3H]uridine at 5 h after infection ("early") or 15 h after infection ("late"), or with 500 jACi of [3H]thymidine for 1 h at 13.5 h after infection ("late"). The rate of viral RNA or DNA synthesis was determined by hybridization as described in the text. b Rate of DNA or RNA synthesis expressed as percentage of ethanol-precipitable radioactivity hybridizable to Ad5 DNA. c ND, Not done. a

gene functions, a culture was infected with a high multiplicity (100 to 200 PFU/cell) of wt virus in the presence of araC. The inhibitor was added again at 10 and 20 h after infection to ensure that deamination of the araC (M. Friedman, unpublished data) did not allow DNA replication. Samples were labeled with either [3H]thymidine or [3H]uridine for 1 h at 7 and 25 h after infection. Incorporation of labeled thymidine was inhibited more than 99.5% at both times. Wt late RNA from uninhibited cells competed with hybridization of RNA labeled at either time only 55% (Fig. 3A). Since wt late RNA contains only class II early and late sequences (12), the uncompeted hybridization must be either class I early RNA or new RNA sequences that are not normally synthesized during adenovirus infection. This latter possibility was ruled out by the complete competition of RNA labeled at 25 h in the presence of araC by early wt RNA (results not shown). To determine whether either of the mutants shut off the synthesis of class I early RNA at 41 C, cells were infected with either ts125 or ts149 and labeled with [3H]uridine for 15 to 16 h after infection; whole-cell RNA was extracted, and its hybridization to Ad5 DNA was competed by unlabeled wt late RNA. The pulselabeled "late" RNA from mutant-infected cells at 41 C was competed only 50% by late RNA (Fig. 3B), indicating that both mutants continued to synthesize class I early RNA. Synthesis of late viral RNA. To test for the transcription of late gene sequences, cells were infected with the mutants at 41 C, and the

159

RNA was labeled from 15 to 16 h after infection; hybridization of the extracted labeled RNA to Ad5 DNA was then competed by wt early RNA (Fig. 4A). Wt early RNA completely inhibited hybridization of labeled RNA from both mutants, but only partially inhibited the hybridization of the labeled wt late RNA control. Therefore, synthesis of late RNA could not be detected by this method in cells infected by either ts125 or ts149 at the restrictive temperature. If a small proportion of labeled viral RNA from the mutant infections consisted of late sequences, these sequences could have escaped detection because of the difficulty in saturating filter-bound DNA with the small amount of labeled early viral RNA characteristically synthesized at 41 C (Fig. 1). The competition analysis was therefore performed in reverse, by hybridizing unlabeled RNA from cells infected by the mutants at 41 C to viral DNA and then challenging the preincubated filters with labeled wt late RNA (Fig. 4B). RNA from ts125infected cells competed 30% of the hybridiza-

o

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w

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I

40 20

20~~~~ A

A

0

0

0.5

1.0

1.5

mg RNA FIG. 2. Competition of wt early RNA hybridization by RNA from cells infected with ts125 or ts149 at 41 C. Ad5 DNA filters (0.2 pg of DNAlfilter) were hybridized to increasing amounts of unlabeled RNA from: cells infected for 15 h at 41 C by ts125 (-) or ts149 (A); and cells infected for 7 h at 36 C by Ad5 wt in the presence of araC (early RNA [0]; uninfected cells [0/). The prehybridized filters were then hybridized to a constant amount (41.4 pg, 3.04 x 105 counts/min) of 3H-labeled wt early RNA. One hundred percent hybridization was 1,440 counts min.

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RNA sequences present in a single preparation by comparing the amounts of this RNA required to compete the hybridization of all homologous sequences of labeled early or late RNA. For this comparison, the data shown in Fig. 2 are reproduced in Fig. 4B (dotted lines). The same unlabeled preparations of the competitor RNA were used in both sets of experiments. With ts125 competitor RNA, maximum competition of wt labeled early or late RNA was

100

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A 75

2

0-_ loo rB >

75

50 0

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25-

00 0

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Ag LATE

200

300

RNA

FIG. 3. Measurement of class I early RNA by hybridization competition. (A) A spinner culture was infected with 100 to 200 PFU of Ad5 wt per cell at 36 C. araC (50 uM/ml) was added at 1, 10, and 20 h after infection. Aliquots were labeled with 5 ,uCi of [3H]uridine per ml from 7 to 8 h after infection (0) or 24 to 25 h after infection (A). Labeled RNA was extracted and competed by wt late RNA. One hundred percent hybridization was 1,820 counts/min for 7- to 8-h RNA (0), and 1,928 counts/min for late RNA (A). (B) Spinner cultures were infected at 41 C with 20 PFU of ts125 (*) or ts149 (A) per cell. RNA was labeled with 5 uCi of [3H]uridine per ml 14 to 15 h after infection, extracted, and competed by wt late RNA. Also competed were labeled wt early RNA (0) and wt late RNA (A). One hundred percent hybridization was 570 counts/min for ts125 DNA; 400 counts/min for ts149 DNA; 580 counts min for early DNA; 1,445 counts/min for late DNA (2 pg of DNAlfilter in experiments summarized in panels A and B).

tion of 3H-labeled late RNA, about the same amount that was competed by unlabeled wt early RNA in the experiment shown in Fig. 4A. However, RNA from cells infected by ts149 competed substantially more wt late RNA hybridization than expected if ts149 RNA consisted only of early sequences. It is possible to estimate the relative amounts of early and late

0

0.5

.I

MG COMPETING RNA FIG. 4. Detection of late viral RNA by competition hybridization. (A) Competition of ts125 and ts149 RNA hybridization by wt early RNA. Spinner cultures were infected and pulse labeled as described in the legend to Fig. 3B. Hybridization of the labeled RNA was competed by wt early RNA from cells infected in the presence of 2 x 10-6 M 5-fluorodeoxyuridine for 6 h (12). Symbols: *, ts125 (100% hybridization, 60 counts/min); A, ts149 (100% hybridization, 99 counts/min); O. wt early RNA (araC) (100% hybridization., 105 counts/min); A, wt late RNA (100% hybridization, 1,538 counts/min). (B) Competition of labeled wt RNA hybridization (100% hybridization, 1,918 counts/min) by unlabeled mutant RNA. Filters containing 0.2 pg of Ad5 DNA were preincubated with increasing amounts of unlabeled RNA from cells infected at 41 C with ts125 (a) or ts149 (A) and then incubated with a constant amount of labeled wt late RNA (solid lines) or labeled wt early RNA (dotted lines).

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TRANSCRIPTION BY Ad5 ts EARLY MUTANTS

achieved at about the same concentration of competitor, 750 /Ag/0.2 ml. At 250 pg/0.2 ml, competition of both labeled preparations was about 75% of the final value. With ts149 competitor RNAs, however, a much higher concentration was needed to achieve the same amount of competition of late wt RNA as of early wt RNA. Therefore, in ts149-infected cells, late viral RNA sequences were present in at least several-fold lower concentrations than the early virus-specific RNAs. Relationship of viral DNA replication to late transcription. The foregoing results indicate that, within the limits of resolution of the filter hybridization technique, both ts125 and ts149 are unable to make a normal transition from early to late transcription at the restrictive temperature. Since neither mutant can synthesize viral DNA under these conditions, each presumably for a different functional reason, the mutants are potentially useful for studying the relationship between viral DNA replication and late gene transcription. The rate of viral DNA synthesis after a shift from 32 C to 41 C at 25.5 h after infection is presented as a function of time in Fig. 5. The rate of synthesis in ts125-infected cells was reduced more than 50% within the first 15 min after a change to the restrictive temperature, and incorporation of thymidine into viral DNA was not detected 100 min after the shift. In contrast, the rate of viral DNA synthesis in ts149-infected cells remained unchanged for 30 min after the shift and decreased slowly thereafter. Even 5 h after the temperature increase, viral DNA synthesis was still detected in ts149infected cells. The temperature shift had no effect on the kinetics of viral DNA synthesis in a wt infection (Fig. 5). To determine the effect of inhibition of DNA replication on viral transcription, the rate of virus-specific RNA synthesis was measured during the same experiments illustrated in Fig. 5. The rate of transcription remained constant in ts125-infected cells for at least 3 h (Fig. 6), whereas in ts149-infected cells the rate of viral RNA synthesis continued to increase at a similar rate to that in wt-infected cells. Thus, with ts125 as well as with wt virus, the rate of transcription was correlated with the amount of DNA replication after the temperature shift. However, viral transcription in ts149-infected cells appeared to be transiently stimulated relative to the rate of viral DNA replication after the shift. To ascertain whether continuing viral DNA replication is necessary for transcription of late viral genes, hybridization competition experiments were done with labeled RNA from cells

161

Z 102

5; o 8IN

04-

0

0-J0

0

2

3

4

5

HOURS AFTER SHIFT FIG. 5. Rate of viral DNA synthesis after a shift from infection of 32 C with 20 PFUlcell of ts125 (@) in one experiment and Ad5 wt (A) or ts149 (A) in a separate experiment. At 25.5 h after infection the temperature was shifted rapidly to 41 C by immersing the spinner flasks in a 60 C water bath and subsequently in a 41 C bath. Samples (50 ml) were labeled for 15 min with [3H]thymidine (250 pXi for wt and ts149, 150 pXi for ts125) at 41 C, except that the first sample from the culture infected with ts125 (zero time) was labeled at 32 C at the time of the shift. DNA was extracted and the percentage of label that hybridized to Ad5 DNA was determined as described in the text.

infected by ts125 at 32 C and shifted to 41 C after DNA synthesis had begun. Cells were infected at 32 C for 26 h, after which half the cells were shifted to the restrictive temperature. The cells at 32 C were labeled with [3H]uridine for 1 h and then harvested. The 41 C culture was labeled from 3.5 to 4.5 h after the temperature shift, at which time no viral DNA synthesis was detected in ts125-infected cells (see Fig. 5). RNA was extracted from the labeled cultures, and the proportion of early and late viral sequences was determined by sequential competition hybridization with DNA in excess relative to labeled viral RNA (Fig. 7). When DNA is in excess, the different labeled viral RNA species will contribute to the total hybridization in proportion to their abundances

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RNA competed with the hybridization of mutant viral RNA labeled before and after the shift to exactly the same extent as labeled wt late RNA (Fig. 7A). Therefore the relative ViJ rates of synthesis of class II early and late RNA I3 sequences were unchanged by the temperature shift. Unlabeled wt late RNA competed all of the hybridization of the RNA from both shifted 41 and unshifted cultures (Fig. 7B), indicating that the majority of the viral RNA species synthesized in each culture was homologous to late RNA. To test for the presence of a small amount of labeled class I early viral RNA, the competition by wt late RNA was done by using 5- to 10-fold more labeled RNA and one-tenth the U. 2 2 3 4 amount of DNA to present labeled RNA in excess of saturation (Fig. 8). Under these conditions, wt late RNA again completely inhibited the hybridization of labeled RNA from cultures both at 32 C and shifted to 41 C 2 3 4 0 5 26 h after infection, indicating that transcripHOURS AFTER SHIFT tion of class I early virus-specific RNA was shut FIG. 6. Rate of viral RNA synthesis after a shift off by 26 h after infection, and that the from 32 to 41 C. The experimental protocol was synthesis of this RNA did not resume after identical to that in Fig. 5, except that 250 ,uCi of DNA replication was inhibited by a shift to the [3H]uridine was used to label samples of the same restrictive temperature. Thus, the virus-specific cultures used for the experiment in Fig. 5. RNA was RNAs made in ts125-infected cells after a shift extracted and the percentage of label that hybridized from 32 to 41 C could not be distinguished from to Ad5 DNA was determined as described in the text. Results are expressed as the rate of viral RNA syn- wt late RNA, in terms both of sequences thesis (percentage of counts per minute incorporated transcribed and the relative amounts of those into viral RNA) relative to the rate at the time of sequences. These data indicate that replicating the shift. The initial rates were determined by DNA is not required for late transcription to labeling at 41 C for wt and ts149 and at 32 C for continue normally in adenovirus-infected cells ts125 (hence, the zero and 20-min points for ts125 are once viral DNA replication has begun. Cl)

z Cl)

z LL

not connected). Initial rates of viral RNA synthesis, expressed as percentage of input counts per minute hybridized to viral DNA, were 0.57 (wt), 0.40 (ts149), and 2.5 (ts125). Symbols: A, wt; 0, ts125; A, ts149.

DISCUSSION Transcription of the adenovirus genome early after infection differs both quantitatively and qualitatively from late viral transcription. (3, 14). Thus, if the rate of transcription of late The results of experiments reported here help RNA sequences were selectively reduced after to define the role played by the viral DNA the temperature shift in ts125-infected cells, wt template in regulating these changes in gene early RNA would compete with a greater activity: (i) the rate of virus-specific RNA synpercentage of the hybridization of RNA labeled thesis both early and late after infection is after the temperature shift than before the dependent upon the amount of viral DNA in shift. The quantities of labeled RNA required the cell; (ii) the onset of viral DNA replication to saturate 0.5 ,ug of viral DNA are shown in is required for the transcription of late genes the insert in Fig. 7A. For DNA excess, and for the shutoff of some early transcription; however, 0.5 jig of DNA was fixed to the filter (iii) neither replicating DNA nor DNA interand the concentrations of labeled RNA used mediates are obligatory templates for late viral were less than those that saturated 0.05 ug of transcription. DNA (insert, Fig. 7A). For saturating condiThe quantitative differences in early and tions, i.e., with labeled viral RNA in excess of late transcription may result largely from an DNA, 0.05 ug of viral DNA was used, and the increase in the amount of viral DNA template amount of labeled RNA was sufficient to obtain present in the infected cells as a result of viral maximum hybridization in uncompeted reac- DNA replication. Before replication, the rate of tions. viral transcription depends upon the number of Under the conditions of DNA excess, wt early infecting genomes (Table 1). During DNA

VOL. 18, 1976

100

A

1000 ujA

~0

75 WN z 0 75

X '] 5.00

4-

100 ug

?00

RNA/50,.&

N 0

50

-

'A

crg

I om

25-

0

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TRANSCRIPTION BY Ad5 ts EARLY MUTANTS

1

2 3 4

5 6

replication, the rate of transcription appears to reflect the number of viral DNA copies that have been produced (Fig. 5 and 6): at late times during wt infection, the rates of viral DNA and RNA synthesis increase in parallel, but a rapid cessation of viral DNA synthesis, after a late shift to 41 C during ts125 infection, effects a constant rate of viral RNA synthesis. Results similar to those for ts125 were obtained when a culture was infected by wt virus and either araC or 5-fluorodeoxyuridine was added after DNA replication had begun (unpublished data; J. J. Lucas, Ph.D. thesis, Univ. of Pennsylvania, Philadelphia, 1972). The result of the temperature shift experiment with ts149 was somewhat different, perhaps because DNA synthesis continued for

7 8

100

mg/ml EARLY WT RNA 100 z

z

0

0

75

75

N N

50

50

cr

m 0

c-T I om

I 0T

25

0

1 2 3 4 mg /mI

5

6 7 8

LATE WT RNA

FIG. 7. Determination of the relative abundances of late RNA synthesized before and after a shift from 32 to 41 C. KB cells (3 liters) were infected at 32 C with 20 PFU of ts125 per cell. At 26 h postinfection, one-half of the culture was labeled with 5 ,uCi of[3H]uridine per ml for 1 h. The other one-half was shifted to 41 C and labeled for 1 h, starting 3.5 h after the shift. Cells were harvested immediately after labeling and RNA was extracted as described in the text. Early wt RNA was labeled from 2 to 7 h postinfection in the presence of araC, and late wt RNA was labeled from 15 to 20 h postinfection. (A) Early wt RNA was prepared from cells infected for 9 h in the presence of araC and was used to compete labeled wt RNA or labeled ts125 RNA from cultures incubated at 32 or 41 C. Viral DNA (0.5 pg) was immobilized on the filters. Labeled RNA (50 pil) was hybridized, after incubation with competitor RNA, as follows: ts125 RNA (0.83 mg/ml) labeled at 32 C

25

O0

0

2

3

mg/ml LATE WT. RNA FIG. 8. Assay for early class I RNA synthesis

after a shift from 32 to 41 C. Late wt RNA was used to compete with the same labeled RNA preparations used in Fig. 7. Filters contained 0.05 pg of viral DNA, and 50 p. of labeled RNA was hybridized at the following saturating concentrations: ts125 (4.1 mg/ml), 32 C (0); ts125 (4.0 mglml), 41 C (A); early wt RNA (3.0 mg/ml) (0); late wt RNA (2.4 mglml) (A).

(0); ts125 (0.80 mg/ml) labeled after the shift to 41 C (A); wt early RNA (0.30 mg/ml) (0); wt late RNA (0.26 mg/ml) (A). (B) Late wt RNA was used to compete with labeled RNA under the same conditions described in (A). Insert: Saturation of 0.05 pg of adenovirus DNA by wt early, wt late, and ts125 RNAs. Symbols are as in (A).

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at least 6 h after the shift, albeit at a decreasing rate (Fig. 5). The increasing rate of viral RNA synthesis during the first 4 h after the shift suggests that continued DNA replication in ts149 transiently stimulated viral transcription on the available templates or increased the number of viral DNA molecules that could be used for transcription. Resolution of this question requires further experiments. The explanation, however, cannot involve the accumulation of replicating DNA intermediates, which might serve as preferred templates for late transcription, because studies show that ts149 completes the replication of viral DNA molecules and does not reinititate replication of most molecules after a shift to the restrictive temperature late in infection (10; M. J. Ensinger, T. Schutzbank, T. H. Carter, and H. S. Ginsberg, in preparation). The qualitative differences between the rates of early and late adenovirus transcription must be the result of a minimum of two specific regulatory events. First, the transcription of late viral genes requires at least that viral DNA replication commence. With two temperature-sensitive DNA-minus mutants from different complementation groups, little or no synthesis of late RNA occurred at the restrictive temperature (Fig. 4), although all normal early sequences were transcribed in amounts comparable to wt (Fig. 1 and 2). This result agrees with experiments in which both viral DNA synthesis and late viral transcription were prevented by inhibitors of DNA replication (1, 5, 12, 14). The small amount of late viral RNA in ts149-infected cells at 41 C could have been the result of viral DNA replication in a small number of cells. If viral DNA synthesis occurred in 0.5% of the population, which was about the limit of detection in these experiments (Table 1), then the overall rate of late viral RNA synthesis in the culture would also be 0.5% of late RNA synthesis in wt-infected cells. Because late RNA is made at nearly 300 times the rate of early RNA in wt infection (Fig. 1), the amounts of early and late RNA should be roughly equal in a population of cells in which only 0.5% are synthesizing viral DNA. In fact, the amount of late viral RNA present in ts149-infected cells at 41 C (and thus the rate of late RNA synthesis, assuming that all viral RNA species are equally stable) was much less than the amount of early RNA (Fig. 4B). Therefore, the present methods do not permit a differentiation between a small amount of viral DNA replication and a weak uncoupling of late transcription from DNA replication with ts149 at 41 C. However, the

J. VIROL.

inability of ts149 to shut off class I RNA synthesis at the restrictive temperature (see below) argues against uncoupling. A second specific regulatory event in the transcriptional program of adenovirus is the shutoff of some early genes late in infection (class I early genes). Ordinarily, the synthesis of class I early RNA begins to be shut off at about the same time that DNA replication is initiated (Lucas, Ph.D. thesis), and by the time late RNA synthesis attains its maximum rate, class I early RNA sequences can no longer be detected by the hybridization competition technique (Fig. 3A and references 6 and 12), DNA saturation analysis (5), or hybridization to restriction endonuclease fragments (H. J. Raskas, personal communication). The failure of a high multiplicity of wt virus to shut off class I RNA synthesis after 25 h in the presence of araC (Fig. 3A) strongly suggests that shutoff is a function of late gene action. Fifteen hours after infection with ts125 or ts149, cells continue to synthesize class I RNA (Fig. 3B). These data, together with the inability of either mutant to synthesize significant amounts of late RNA at 41 C (Fig. 4), indicate that neither of the DNA-minus mutants is able to make the normal transition from early to late gene expression at the restrictive temperature. Although it is not yet possible to identify the nature of the regulatory events that result in the switch from early to late transcription, the temperature shift experiment with ts125 indicates that concomitant viral DNA replication is not required for the transcription of late genes. When ts125 DNA synthesis was completely inhibited by a shift from 32 to 41 C late in infection, the late mode of transcription was maintained (Fig. 7 and 8). This result considerably strengthens the same conclusion drawn from earlier experiments in which araC or 5-fluorodeoxyuridine inhibited viral DNA replication but failed to prevent late transcription once the synthesis of viral DNA had ensued (Lucas, Ph.D. thesis). In the light of evidence suggesting that ts125, like ts149, is temperature sensitive for the initiation of viral DNA replication (10; Ensinger et al., manuscript in preparation), the continued synthesis of late ts125-specific RNA 3 to 4 h after a shift from 32 to 41 C implies that DNA replicative intermediates, which progressively and rapidly disappear after the shift, are not required for late viral transcription. It is possible, however, that a relatively stable pool of single-stranded DNA, which might be generated by displacement of both DNA strands during replication,

TRANSCRIPTION BY Ad5 ts EARLY MUTANTS

VOL. 18, 1976

could escape detection by the usual methods of isopycnic density gradient and velocity sedimentation (11). If late transcription requires single-stranded regions of the DNA, this potential phenomenon might account for continued synthesis of late viral RNA after a temperature shift in ts125-infected cells. Further experiments will be needed to test this possibility. The conclusion that replicative intermediates in viral DNA synthesis are not obligatory templates for late viral transcription was also reached for simian virus 40 by similar temperature shift experiments with a tsA early mutant that is defective in initiation of DNA replication (4). However, the results obtained with the tsA mutant did not rule out the possibility that the rate of simian virus 40 transcription was reduced in the absence of viral DNA replication after the shift. In fact, recent experiments in which simian virus 40 DNA replication was inhibited by Miracil D, which prevents initiation of viral DNA synthesis, showed that the rate of viral RNA transcription was much reduced (13). In the experiments reported here with temperature-sensitive adenovirus mutants, however, it is shown that neither the overall rate of viral transcription nor the relative amounts of class II early and late transcripts are altered in the absence of replicating DNA. If the physical state of the DNA template does play a crucial role in the regulation of adenovirus transcription, this part must be more subtle than a requirement for replicating DNA molecules. Perhaps template regulation initially involves the masking of late gene sequences or promoters by viral core proteins. Another possibility is that late gene transcription is activated by a function associated with the initiation or termination steps of DNA replication, as has been postulated for the bacteriophage T4 (20). In the case of adenovirus, however, this activation appears to be irreversible. Such an activation of late gene transcription might be effected by a regulatory protein whose expression requires only the commencement of viral DNA replication. Further studies with the temperature-sensitive, DNA-minus mutants of adenovirus are being done to test this hypothesis. ACKNOWLEDGEMENTS It is a pleasure to acknowledge the excellent assistance of J. Higgs and P. Muntz in the culture of cells. Thanks are also extended to G. Zubay for providing Xdlac DNA, to A. Mayer for helpful discussions related to DNA:DNA hybridization techniques, and to M. Ensinger and T. Schutzbank for permission to quote unpublished results

165

pertaining to the initiation of DNA replication by the DNA-minus mutants. This research was supported in part by Public Health Service grants AI-12052 and AI-12053 from the National Institute of Allergy and Infectious Diseases and by the U.S. Army Medical Research Development Command, Department of the Army, under research contract DADA-1773-3153. T.H.C. was a recipient of postdoctoral fellowships from the William J. Matheson Foundation and the National Cancer Institute (Public Health Service grant 5 F22 CA02751).

APPENDIX Assay of viral DNA by filter hybridization. If hybridization of pulse-labeled DNA to viral DNA immobilized on membrane filters is used to measure the rate of synthesis of viral DNA in infected cells, the method suffers the inherent drawback that the self-annealing of the DNA in solution competes with the hybridization to DNA on the filter. Thus, direct hybridization always underestimates the real amount of labeled DNA in the sample. This problem, which is particularly bothersome when pulse-labeled viral DNA comprises a variable fraction of the viral DNA in the sample, can, in principle, be overcome if a large excess of filter-bound DNA is used. However, it is often impractical to use the amount of immobilized DNA that would be necessary to offset completely the self-annealing of the viral DNA in solution. We have used an alternative procedure that measures the efficiency of hybridization of purified viral DNA that has been added to the sample containing unknown amounts of labeled and unlabeled viral DNA to account for self-annealing. Because the amount of self-annealing in solution should be dependent upon the total concentration of viral DNA, hybridization efficiencies of the purified "standard" DNA and the pulse-labeled "unknown" viral DNA should be the same. To test the validity of this method, a reconstruction experiment was done in which a constant amount of purified, 14C-labeled adenovirus DNA was added to hybridization reactions containing increasing amounts of purified 3H-labeled viral DNA in place of the "unknown." The hybridizations of both isotopes to filters containing denatured viral DNA are plotted in Fig. 9A. As the amount of 3Hlabeled viral DNA was increased, hybridization of 14C correspondingly decreased, and the hybridization of 3H deviated increasingly from linearity with respect to input. However, when the efficiency of hybridization of the '4C was calculated for each point (counts per minute hybridized/counts per minute input) and this efficiency was used to correct the measured 3H hybridization, a linear relationship between 3H input and hybridization was obtained (Fig. 9B). When a similar reconstruction experiment was done using increasing amounts of pulse-labeled DNA extracted from infected cells, a linear relationship between input and hybridization was also obtained (results not shown). The slope of the corrected 3H hybridization in Fig. 9B was identical to that predicted for 100% hybridization, suggesting that the intrinsic efficiency of hybridization of the two DNA preparations was the same.

J. VIROL.

CARTER AND GINSBERG

166 0

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ity of early and late RNA. J. Mol. Biol. 31:325-348. 4. Cowan, K., P. Tegtmeyer, and D. D. Anthony. 1973. U&.

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(specific activity, 17,800 with 0.44 counts/mm per ig) were of puri"iC-labeled adenovirus DNA (specific activity, 11,300 per pg) and 200 pg of DNA in a total volume of 1.5 ml. The mixtures were to 5 denatured and ug of DNA immobilized on membrane filters as described and in text. (A) Hybridizations of ['4C]DNA (A) are plotted as a function of[PH]DNA input. Efficiency of hybridization of [14C]DNA (counts per minute hybridized/counts per minute ranged from 0.45 to less than 0.25. (B) Hybridization of 3H-labeled viral DNA from (A) was corrected by dividing each value by the calculated efficiency of "IC hybridization in each sample (0). The line drawn is the calculated result if the 3H hybridization were 100% efficient.

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LITERATURE 1.

16. 17.

CITED

Bello, L. J., and H. S. Ginsberg. 1969. Relationship

between deoxyribonucleic acid-like ribonucleic acid synthesis and inhibition of host protein synthesis in type 5 adenovirus-infected KB cells. J. Virol. 3:106113. 2. Bock, R. M. 1967. Controlled partial hydrolysis of RNA, p. 218-224. In L. Grossman and K. Moldave (ed.), Methods in enzymology, vol. 12A. Academic

Press Inc., New York. 3. Bolle, A., R. H. Epstein, W. Salser, and E. P. Geiduschek. 1968. Transcription during bacteriophage T4 development: synthesis and relative stabil-

18.

Relationship of replication and transcription of simian virus 40 DNA. Proc. Natl. Acad. Sci. U.S.A.. 70:1927-1930. Craig, E. A., and H. J. Raskas. 1974. Effect of cycloheximide on RNA metabolism early in productive infection with adenovirus 2. J. Virol. 14:26-32. Craig, E. A., and H. J. Raskas. 1974. Two classes of cytoplasmic viral RNA synthesized early in productive infection with adenovirus 2. J. Virol. 14:751757. Ensinger, M. J., and H. S. Ginsberg. 1972. Selection and preliminary characterization of temperaturesensitive mutants of type 5 adenovirus. J. Virol. 10:328-339. Friedman, M. P., M. J. Lyons, and H. S. Ginsberg. 1970. Biochemical consequences of type 2 adenovirus and simian virus 40 double infections of African green monkey kidney cells. J. Virol. 5:586-597. Fujinaga, K., and M. Green. 1970. Mechanism of viral carcinogenesis by DNA mammalian viruses. VII. Viral genes transcribed in adenovirus 2 infected and transformed cells. Proc. Natl. Acad. Sci. U.S.A. 65:375-382. Ginsberg, H. S., M. J. Ensinger, R. S. Kauffman, A. J. Mayer, and U. Lundholm. 1970. A study of regulation with types 5 and 12 adenovirus temperaturesensitive mutants. Cold Spring Harbor Symp. Quant. Biol. 39:419-426. Lavelle, G., C. Patch, G. Khoury, and J. Rose. 1975. Isolation and partial characterization of singlestranded adenoviral DNA produced during synthesis of adenovirus type 2 DNA. J. Virol. 16:775-782. Lucas, J. J., and H. S. Ginsberg. 1971. Synthesis of virus-specific ribonucleic acid in KB cells infected with type 2 adenovirus. J. Virol. 8:203-213. Manteuil, S., and M. Girard. 1974. Inhibitors of DNA synthesis: their influence on replication and transcription of simian virus 40 DNA. Virology 60:438454. Parsons, J. T., and M. Green. 1971. Biochemical studies of adenovirus multiplication. XVIII. Resolution of early virus-specific RNA species in Ad2 infected and transformed cells. Virology 45:154-162. Salser, W., A. Bolle, and R. Epstein. 1970. Transcription during bacteriophage T4 development: a demonstration that distinct subclasses of the "early" RNA appear at different times and that some are "turned off" at late times. J. Mol. Biol. 49:271-295. Sutton, W. D. 1971. A crude nuclease preparation suitable for use in DNA reassociation experiments. Biochim. Biophys. Acta 240:522-531. Thomas, D. C., and M. Green. 1969. Biochemical studies on adenovirus multiplication. XV. Transcription of the adenovirus type 2 genome during productive infection. Virology 39:205-210. Tibbetts, C., K. Johansson, and L. Philipson. 1973. Hydroxyapatite chromatography and formamide denaturation of adenovirus DNA. J. Virol. 12:218-

225. 19. Van der Eb, A. J. 1973. Intermediates in type 5 adenovirus DNA replication. Virology 5:11-23. 20. Wu, R., E. P. Geiduscheck, D. Rabussay, and A. Cascino. 1973. Regulation of transcription in bacteriophage T4-infected E. coli-a brief review and some recent results, p. 181-199. In C. F. Fox and W. S. Robinson (ed.), Virus research. Academic Press Inc., New York.